Stable emulsions of highly fluorinated organic compounds

ABSTRACT

Stable emulsions of highly fluorinated organic compounds for use as oxygen transport agents, &#34;artificial bloods&#34; or red blood cell substitutes and as contrast agents for biological imaging. The emulsions comprise a highly fluorinated organic compound, an oil that is not substantially surface active and not significantly soluble in water, a surfactant and water.

This application is a continuation of application Ser. No. 06/883,713,filed Jul. 9, 1986 and now abandoned.

TECHNICAL FIELD OF INVENTION

This invention relates to stable emulsions of highly fluorinated organiccompounds and to processes of making and using them. More particularly,this invention relates to novel emulsions, stable even at roomtemperature, that comprise an oil, that is not substantially surfaceactive and not significantly soluble in water, water, a surfactant and ahighly fluorinated organic compound. Such emulsions are especiallyuseful in compositions for use as oxygen transport agents, "artificialbloods" or red blood cell substitutes and as contrast agents forbiological imaging.

BACKGROUND OF THE INVENTION

Highly fluorinated organic compounds are well known to be chemically andpharmaceutically inert and to be capable of dissolving and transportinglarge amounts of oxygen. These properties make them potentially usefulas oxygen transport agents, "artificial bloods" or red blood cellsubstitutes and as contrast agents for various imaging modalities, suchas nuclear magnetic resonance, ultrasound, and x-ray. However, neatfluorocarbon liquids cannot be injected into the blood stream, becausetheir hydrophobic character makes them immiscible in the blood and as aresult, when they are transported into small blood vessels they maycause vascular obstruction and death. As a consequence, for medical usesthat require intravascular injection, highly fluorinated organiccompounds must be dispersed as physiologically acceptable emulsions.See, e.g., L. C. Clark, Jr. et al., "Emulsions Of PerfluorinatedSolvents For Intravascular Gas Transport", Fed. Proc., 34 (6), pp.1468-77 (1975); K. Yokoyama et al., "A Perfluorochemical Emulsion As AnOxygen Carrier", Artif. Organs (Cleve), 8 (1), pp. 34-40 (1984); andU.S. Pat. Nos. 4,110,474 and 4,187,252.

To date, however, the medical usefulness of such emulsions of highlyfluorinated organic compounds as "artificial bloods" or bloodsubstitutes, oxygen transport agents or contrast agents for biologicalimaging has not been as successful as hoped. This results from the factthat in practice it has not been previously possible to make emulsionsthat are both stable and incorporate the relatively large amounts ofhighly fluorinated organic compounds that are required in clinicalpractice where the total volume of emulsion that can be administered islimited, e.g., as "artificial bloods". Moreover, it has not beenpreviously possible to make such emulsions using highly fluorinatedorganic compounds that are excreted from the body within a clinicallyacceptable time period (see U.S. Pat. No. 3,911,138). Finally, eventhose, admittedly less than therapeutically acceptable, compositionsthat have been available to date are difficult to sterilize because oftheir instability at high temperature.

Various attempts have been made to solve these problems and to preparestable emulsions containing high concentrations of clinically suitablehighly fluorinated organic compounds. None has been successful. Forexample, a variety of fluorocarbons and combinations of them have beenused in preparing the emulsions in hopes of improving their stability.None has produced a medically effective and commercially acceptableemulsion that is stable at room temperature. For example, the onlyfluorocarbon emulsion to reach clinical testing as an "artificialblood", "Fluosol DA 20%" is about a 12% by volume emulsion of twofluorocarbons--perfluorodecalin and perfluorotripropylamine--in amixture of two surfactants--yolk phospholipid and Pluronic F-68. It isnot stable in the liquid state and must be stored frozen (Yokoyama etal., supra). Furthermore, the required presence of theperfluorotripropylamine in this emulsion, to help "stabilize" it,disadvantages the emulsion's medical usefulness because the half-life ofthe perfluorotripropylamine in the liver and other body tissues islonger than desirable (see, e.g., K. Yokoyama et al., supra). Finally,because this emulsion contains only about 12% fluorocarbon by volume, itis much less therapeutically effective than desired because of its lowoxygen content capacity (see, e.g., "Fluosol-DA As A Red Cell SubstituteIn Acute Anemia", N.E. Jour. Med., 314, pp. 1653-66 (1986).

Emulsions of other perfluorocarbons have likewise not been veryeffective in avoiding these instability and oxygen capacity problems.For example, an emulsion of perfluoro-4-methyloctahydroquinolidizine(FMOQ) in two surfactants--Pluronic F-68 and yolk phospholipid--must bestored at 4° C. (K. Yokoyama et al., supra).

Various surfactants have also been investigated in the hope that somewould produce useful, stable emulsions of highly fluorinated organiccompounds for use as oxygen transport agents and "artificial bloods".Again, these attempts have failed. For example, fluorocarbon emulsionscontaining a hydrogenated phospholipid, a nonionic polymeric surfactantand a surfactant selected from 6-22 C fatty acids, their salts andmonoglycerides must also be stored at 4° C. See, e.g., Japanese patentapplication 59,067,229, U.S. Pat. No. 4,252,827 and Germany Offen. DE2630506.

Therefore, the medical and non-medical uses of highly fluorinatedorganic compounds as effective oxygen transport agents, "artificialbloods" or red blood cell substitutes, and contrast agents forbiological imaging is still a long sought and important goal.

SUMMARY OF THE INVENTION

This invention solves the problems referred to above. This inventionprovides novel emulsions of highly fluorinated organic compounds for useas oxygen transport agents, "artificial bloods" or red blood cellsubstitutes, and as contrast agents for various biological imagingmodalities. This invention also provides emulsions that are stable evenwhen they contain the higher levels of highly fluorinated organiccompounds that are required in emulsions for use as blood substitutesbecause of the high oxygen content capacity required in thatapplication. This invention also provides stable emulsions that employonly those fluorocarbons that display acceptably rapid excretion timesfrom the liver and other body tissues. Finally, this invention providesemulsions that are easily sterilized.

The emulsions of this invention comprise at least one highly fluorinatedorganic compound; an oil that is not substantially surface active andnot significantly soluble in water; a surfactant and water.

This invention also includes methods of making these emulsions andmethods and compositions of using them as oxygen transport agents,"artificial bloods" or red blood cell substitutes, and contrast agentsfor biological imaging.

DETAILED DESCRIPTION OF THE INVENTION

The emulsions of this invention comprise at least one highly fluorinatedorganic compound; an oil that is not substantially surface active andnot significantly soluble in water; a surfactant and water.

The preferred emulsions of this invention are stable at room temperaturefor long periods of time. They exhibit substantially no phase separationand substantially no change in particle or droplet size distributionduring storage. Moreover, they permit the use of highly fluorinatedorganic compounds that exhibit acceptably rapid excretion times from theliver and other body tissues. And, they permit the use of the highconcentrations of fluorocarbons thereby producing the high oxygencontent capacity emulsions required for use of the emulsions of thisinvention as therapeutically effective blood substitutes. Finally,because of their stability, the emulsions of this invention may besterilized by heating them to high temperature, for example, 115° C. for15 min. It is one very surprising aspect of this invention that evensuch harsh conditions do not cause phase separation of the emulsions ofthis invention. As a result of these novel and unexpected properties,the emulsions of this invention solve the long standing problems ofprior fluorocarbon-containing compositions and make these highlyfluorinated organic compounds available for the first time incommercially useful forms for use as oxygen transport agents,"artificial bloods" or red blood cell substitutes and as contrast agentsfor biological imaging.

While not wishing to be bound by theory, we believe that the emulsionsof this invention may have the highly fluorinated organic compounddispersed in oil and that oil-fluorocarbon combination emulsified in thewater and surfactant. However, other possible phases and interfaces arealso within the scope and intent of this invention.

Among the highly fluorinated organic compounds that are useful in theemulsions and processes of this invention are those previously said tobe useful as oxygen transport agents, "artificial bloods" or red bloodcell substitutes, and contrast agents for biological imaging. Theseinclude, for example, perfluorocarbons, partially fluorinatedhydrocarbons and derivatives and mixtures of them. For example, amongthe fluoro-containing compounds useful in the emulsions of thisinvention are 9-18C perfluorohydrocarbons, e.g., perfluorodecalin,perfluoro-trimethyl-bicyclo 3.3.1! nonane andperfluoro-2,2,4,4-tetramethylpentane, 9-12C perfluoroamines, e.g.,perfluorotripropylamine, perfluorotributylamine,perfluorodimethyladamantane, perfluoro-1-aza-tricylic amines, bromo- oriodo-substituted fluorocarbons, and F-4-methyloctahydroquinolidizine.Such compounds are described, for example, in U.S. Pat. Nos. 3,962,439,3,493,581, 4,110,474, 4,186,253, 4,187,252, 4,252,827, 4,423,077,4,443,480, 4,534,978 and 4,542,147, European patent applications 80710and 158,996, British patent specification 1,549,038 and German Offen.2,650,586. Of course, it should be understood that mixtures of any ofthese highly fluorinated organic compounds may also be used in theemulsions and processes of this invention.

Preferably, the emulsions of this invention contain one or more of aperfluorocarbon and most preferably a fluorocarbon selected from thegroup consisting of perfluorodecalin, perfluorodimethyladamantane,perfluorooctylbromide, perfluoro-4-methyl-octahydroquinolidizine,perfluoro-N-methyl-decahydroquinoline, F-methyl-1-oxa-decalin,perfluoro-bicyclo 5.3.0! decane, perfluorooctahydroquinolidizine,perfluoro-5,6-dihydro-5-decene, and perfluoro-4, 5-dihydro-4-octene. Foruse as a contrast agent for biological imaging perfluorooctylbromide isone of the preferred highly fluorinated organic compounds according tothis invention.

While the highly fluorinated organic compounds or mixture of suchcompounds may comprise up to about 75% (by volume) of the emulsions ofthis invention. Preferably, the emulsions of this invention comprisefrom 10% to about 70% (by volume) of the fluorocarbon. When theemulsions are to be used as "artificial bloods" or red blood cellsubstitutes, the fluoro-containing compounds are preferably present inas high a volume concentration as possible. However, 40% (by volume) isoften preferred because that concentration matches the approximateoxygen content capacity of whole blood.

Among the not substantially surface active and not significantly watersoluble oils that are useful in the emulsions and processes of thisinvention are liquid fatty oils, hydrocarbons, waxes, such as monoestersof a fatty acid and a monohydroxide alcohol, long chain ethers,diglycerides, silicone oils and nitriles. These include, for example,palmitoyl oleate, octyl nitrile, dodecyl nitrile, triglycerides of fattyacids such as soy oil, and safflower oil, hexadecane, diglycerideshaving a C₁₂₋₁₈ carbon chain and one unsaturation, and mineral oil. Aswith the fluoro-containing component, these oils also may be used singlyor in various combinations in the emulsions and processes of thisinvention. When our emulsions are to be used medically, the oil orcombination of oils must, of course, be physiologically acceptable. Forexample, when our emulsions are to be used as "artificial bloods", wepreferably use physiologically acceptable liquid fatty oils.

The amount of oil, or oils, present in the emulsions of this inventionmay vary over a wide range of concentrations. It depends on theconcentration and properties of the other components of the emulsion,being principally dependent on the characteristics of the fluorocarboncomponent of the emulsion. The actual oil concentration to produce anacceptable emulsion for any given set of components is easily determinedas taught by this invention using the simple techniques of preparing andtesting the stability of emulsions at various oil concentrations. Withinthis teaching, we typically employ between about 10 and 30% (by weightof the remaining non-fluorocarbon volume of the emulsion) of oil or amixture of oils. Preferably, we employ between about 15 and 20% byweight.

Among the surfactants useful in the emulsions of this invention are anyof the known anionic, cationic, nonionic and zwitterionic surfactants.These include, for example, anionic surfactants, such as alkyl or arylsulfates, sulfonates, carboxylates or phosphates, cationic surfactantssuch as mono-, di-, tri-, and tetraalkyl or aryl ammonium salts,nonionic surfactants, such as alkyl or aryl compounds, whose hydrophilicpart consists of polyoxyethylene chains, sugar molecules, polyalcoholderivatives or other hydrophilic groups and zwitterionic surfactantsthat may be combinations of the above anionic or cationic groups, andwhose hydrophobic part consists of any other polymer, such aspolyisobutylene or polypropylene oxides. Again, combinations of thesesurfactants may, of course, be used in the emulsions of this invention.In addition, mixtures of compounds, one or more of which are notsurfactants, but which compounds when combined act as surfactants mayalso be usefully employed as the surfactant component of the emulsionsof this invention.

Again, when the emulsions of this invention are to be used in"artificial bloods" or red blood cell substitutes, the surfactant, orcombinations of them, must be physiologically acceptable. For example,in "artificial bloods" we prefer non-ionic surfactants. Preferably, thesurfactants used in the emulsions of this invention are one or more ofthe following: egg phosphatides, lecithin, and alkyl salts of oleicacid, such as sodium oleate.

While the amount of a particular surfactant used in the emulsions ofthis invention depends on the amounts and properties of the othercomponents of the emulsion, typically we employ about 0.5 to 7% (byweight of the non-fluorocarbon volume) of surfactant. More preferably,we use about 1-2% (by weight).

In addition to the highly fluorinated organic compounds, oils,surfactants and water, the emulsions of this invention may also containother components conventionally used in "artificial bloods" or bloodsubstitutes, oxygen transport agents or contrast agents for biologicalimaging. For example, when used as a blood substitute, an emulsionaccording to this invention should contain an isotonic agent, typicallyglycerol, to adjust the osmotic pressure of the emulsion to about thatof blood. Typically we use about 2.5% (by weight of the non-fluorocarbonvolume) of glycerol. However, other amounts and other osmotic pressurecontrolling agents, e.g., Tyrode solution, could as well be used. Theemulsions of this invention may also include other components, such asoncotic agents, e.g., dextran or HES, and antioxidants.

The emulsions of this invention may be prepared using any order ofmixing the four main components of our emulsions--highly fluorinatedorganic compound, oil, surfactant and water. However, for an optimalemulsion we prefer to mix the fluorocarbon first with the oil in thepresence of a combination of all or part of the surfactant and somewater. We then prepare the final emulsion by emulsifying this firstemulsion in the remaining water and any remaining surfactant.

The mixing and emulsification of our components may be done using any ofthe conventional mixers and emulsifiers. For example, we may employFisher brand touch mixers and Microfluidizers. We may also, if desired,reduce the average size of the droplets or particles in our emulsions byconventional grinding.

EXAMPLE 1

In this example, we prepared two emulsions in the same manner to comparetheir stabilities. The first emulsion was a conventional compositioncomprising 40% by volume perfluorodecalin and 60% by volume of a mixtureof water (96.3% by weight), lecithin (1.2% by weight), glycerol (2.5% byweight) and sodium hydroxide to pH 8. The second emulsion was preparedaccording to this invention. It had the following composition: 40% byvolume perfluorodecalin and 60% by volume of a mixture of water (78.8%by weight), lecithin (1.2% by weight), glycerol (2.5% by weight) soy oil(17.5% by weight) and sodium hydroxide to pH 8. The two emulsions wereprepared by mixing their components together in a Fisher brand touchmixer and then running them through a Microfluidizer for 30 min at 60psi.

The first emulsion had a smaller average droplet size by opticalmicroscopy than the second emulsion. It also had a higher concentrationof water and thus less dispersed phase than the second emulsion.Accordingly, on those bases alone, we would have expected the firstemulsion to be more stable than the second emulsion. However, the first"emulsion" was very unstable and exhibited phase separation at roomtemperature within 24 hours. The second emulsion (that prepared inaccordance with this invention), while having a larger average dropletor particle size and more dispersed phase, was surprisingly very stableand showed substantially no phase separation and substantially no changein droplet or particle size distribution during 4 weeks storage at roomtemperature.

This comparison plainly demonstrates that the emulsions of thisinvention are different in kind from former compositions of highlyfluorinated organic compounds. Not only are our emulsions far morestable, they are surprisingly more stable even with larger averageparticle or droplet size and more dispersed phase.

EXAMPLE 2

We prepared an emulsion containing 40% by volume perfluorodecalin and60% by volume of a first emulsion containing safflower oil (10% byweight), soybean oil (10% by weight), lecithin (1.2% by weight),glycerol (2.5% by weight), water (76.3% by weight) and sodium hydroxideto pH 8.3. We prepared the final emulsion by combining 20 mlperfluorodecalin and 30 ml of the first emulsion and mixing thecombination in a Fisher touch-mixer for 20 min. We then ran theresulting emulsion through a Microfluidizer for 1 hour at 60 psi.

The resulting homogenized emulsion was still stable after 4 weeks atroom temperature, as demonstrated by optical microscopy which indicatedthat there had been substantially no change in particle or droplet sizedistribution and substantially no phase separation.

EXAMPLE 3

Using the substantially same process as described in Example 2, weprepared an emulsion containing 40% by volume perfluorodecalin and 60%by volume of a first emulsion containing safflower oil (10% by weight),soybean oil (10% by weight), lecithin (2.0% by weight), glycerol (2.5%by weight), water (75.5% by weight) and sodium hydroxide to pH 8.3. Asbefore, the resulting emulsion was still stable after 4 weeks at roomtemperature.

EXAMPLE 4

We prepared an emulsion containing 40% by volume perfluorodecalin and60% by volume of a first emulsion containing safflower oil (10% byweight), soybean oil (10% by weight), lecithin (2.0% by weight),glycerol (2.5% by weight), XMO-20 (see, e.g., U.S. Pat. No. 4,443,480)(0.1% by weight), water (75.4% by weight) and sodium hydroxide to pH8.3. We prepared the final emulsion by combining 20 ml perfluorodecalinand 30 ml of the first emulsion and mixing the combination in a Fishertouch mixer until the lecithin and XMO-20 were completely dissolved. Wethen ran the emulsion through a Microfluidizer for 30 min at 60 psi. Theresulting emulsion was still stable after 4 weeks at room temperature.

EXAMPLE 5

We prepared an emulsion containing 40% by volume perfluorodecalin and60% by volume of a first emulsion containing safflower oil (10% byweight), soybean oil (10% by weight), lecithin (1.2% by weight),glycerol (2.5% by weight), oleic acid (0.8% by weight), water (75.5% byweight) and sodium hydroxide to pH 8.3. We prepared the final emulsionby mixing 20 ml perfluorodecalin and 30 ml of the first emulsion in aFisher touch mixer for 10 min and running the resulting emulsion througha Microfluidizer for 45 min. The resulting emulsion was still stableafter weeks at room temperature.

EXAMPLE 6

We added lecithin to a final concentration of 2% (by weight) to thefinal emulsion of Example 5 and mixed it until the lecithin hadcompletely dissolved. The emulsion was then run through a Microfluidizerfor 30 min at 60 psi. The resulting emulsion was still stable after 4weeks at room temperature.

EXAMPLE 7

We prepared an emulsion containing 40% by volume perfluorodecalin and60% by volume of a mixture containing water (78.8% by weight), lecithin(1.2% by weight), glycerol (2.5% by weight), soybean oil (17.5% byweight) and sodium hydroxide to pH 8.0. We prepared the emulsion bymixing 0.377 g lecithin and 20 ml perfluorodecalin in a Fisher brandtouch mixer for 10 min. We then added 5.4915 g soybean oil and mixedagain for 10 min and added 0.7945 g glycerol and mixed again for 10 min.Finally, we added 24.721 g water stepwise with mixing. We made thisaddition by first adding 12.36 g of water to the mixture to disperse thefluorocarbon-oil-lecithin mixture and emulsified the resultingdispersion in a Microfluidizer for 30 min at 60 psi. We then emptied theemulsion from the Microfluidizer and poured the remaining water into theMicrofluidizer. After adding the previously prepared emulsion dropwiseto the water, we ran the resulting mixture through the Microfluidizerfor 30 min at 60 psi and adjusted the pH to 8.0 with sodium hydroxide.We then again ran the emulsion through a Microfluidizer for 30 min at 60psi. The final emulsion was still stable after 4 weeks at roomtemperature.

EXAMPLE 8

We prepared an emulsion containing 40% by volume perfluorodecalin and60% by volume of a mixture containing water (78.8% by weight), lecithin(1.2% by weight), glycerol (2.5% by weight), hexadecane (17.5% byweight) and sodium hydroxide to pH 8.0. We used substantially the samemethod described in Example 7. The final emulsion was still stable after4 weeks at room temperature.

EXAMPLE 9

We prepared an emulsion similar to that of Example 8, except thathexadecane was replaced with mineral oil. Again, we used substantiallythe same method described in Example 7 to prepare the emulsion. Thefinal emulsion was still stable after 4 weeks at room temperature.

EXAMPLE 10

We prepared an emulsion containing 55% by volume perfluorodecalin and45% by volume of a mixture containing safflower oil (10% by weight),soybean oil (10% by weight), glycerol (2.5% by weight), lecithin (2% byweight) and water (75.5% by weight) and sodium hydroxide to pH 8. Toprepare the emulsion we used 18 ml of the oil-containing mixture, 22 mlof perfluorodecalin and 1% (by weight on total) oleic acid. We used themethod substantially as described in Example 7. We mixed the finalemulsion for 20 min in a Fisher touch mixer and then in a Microfluidizerfor 15 cycles at 65 psi. The emulsion was stable at room temperature.

EXAMPLE 11

We prepared an emulsion containing 70% by volume perfluorodecalin and30% by volume of a mixture containing safflower oil (10% by weight),soybean oil (10% by weight), lecithin (1.2% by weight), glycerol (2.5%by weight), water (76.3% by weight) and sodium hydroxide to pH 8.0. Toprepare the final emulsion we used 21 ml perfluorodecalin, 9 ml of theoil-containing mixture, and 0.5% (by weight on total) oleic acid. As inExample 10, we used substantially the same method described in Example 7to prepare the final emulsion. We then mixed the final emulsion for 20min in a Fisher touch mixer and then in a Microfluidizer for 15 cyclesat 65 psi. The emulsion was stable at room temperature.

EXAMPLE 12

We prepared an emulsion containing 16.5 ml perfluorooctylbromide (55% byvolume) and 13.5 ml of the same mixture of other components described inExample 11. We used the same mixing and fluidizing regime described inExamples 10 and 11. The final emulsion was stable at room temperature.

While we have hereinbefore described a number of embodiments of ourinvention, it should be apparent that other embodiments also existwithin our invention. Therefore, it should be understood that the scopeof this invention is to be defined by the claims rather than by thespecific embodiments which have been presented hereinbefore by way ofexample.

We claim:
 1. A stable aqueous emulsion of a perfluorochemical comprisingapproximately 60 weight/volume percent or greater of aperfluorochemical, approximately 0.5 up to 7 weight % of a phospholipidwhich emulsifies said perfluorochemical, approximately 5-30 weight % ofa triglyceride of fatty acids, and the remainder of an aqueous medium.2. A stable aqueous emulsion of a perfluorochemical comprisingapproximately 15-70% by volume perfluorochemical, a phospholipid whichemulsifies said perfluorochemical, a triglyceride of fatty acids as anemulsifier adjuvant, and water.
 3. The stable aqueous emulsion of claim2 wherein the emulsion comprises approximately 40-70% by volumeperfluorochemical.
 4. A stable aqueous emulsion of a perfluorochemicalcomprising approximately 15-70% by volume perfluorochemical,approximately 0.5 up to 7 weight % of a phospholipid which emulsifiessaid perfluorochemical, approximately 5-30 weight % of a triglyceride offatty acids as an emulsifier adjuvant, and the remainder of an aqueousmedium.
 5. A stable aqueous emulsion of a perfluorochemical comprisingapproximately 15-70% by volume perfluorochemical, a phospholipid whichemulsifies said perfluorochemical, a triglyceride of fatty acids as anemulsifier adjuvant, and the remainder of an aqueous medium, wherein thephospholipid and triglyceride of fatty acids are present in amountseffective to provide a stable emulsion.
 6. A stable aqueous emulsion ofa perfluorochemical comprising from 10 to about 70% by volumeperfluorochemical, about 0.5 to 7% by weight of the non-fluorocarbonvolume of a phospholipid which emulsifies said perfluorochemical, about10 to 30% by weight of the non-fluorocarbon volume of a triglyceride offatty acids as an emulsifier adjuvant, and water.